Differential innate immune signalling via Ca2+ sensor protein kinases

Innate immunity represents the first line of inducible defence against microbial infection in plants and animals. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively), such as flagellin, initiates convergent signalling pathways involving mitogen-activated protein kinase (MAPK) cascades and global transcriptional changes to boost immunity. Although Ca2+ has long been recognized as an essential and conserved primary mediator in plant defence responses, how Ca2+ signals are sensed and relayed into early MAMP signalling is unknown. Using a functional genomic screen and genome-wide gene expression profiling, here we show that four calcium-dependent protein kinases (CDPKs) are Ca2+-sensor protein kinases critical for transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in the synthesis of defence peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by the 22-amino-acid peptide flg22, as well as compromised pathogen defence. In contrast to negative roles of calmodulin and a calmodulin-activated transcription factor in plant defence, the present study reveals Ca2+ signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.

[1]  A. Huffaker,et al.  Endogenous peptide defense signals in Arabidopsis differentially amplify signaling for the innate immune response , 2007, Proceedings of the National Academy of Sciences.

[2]  Ping He,et al.  Bacterial effectors target the common signaling partner BAK1 to disrupt multiple MAMP receptor-signaling complexes and impede plant immunity. , 2008, Cell host & microbe.

[3]  Wei-Min Liu,et al.  Robust estimators for expression analysis , 2002, Bioinform..

[4]  Yule Liu,et al.  Efficient Virus-Induced Gene Silencing in Arabidopsis1 , 2006, Plant Physiology.

[5]  T. Romeis,et al.  Calcium‐dependent protein kinases play an essential role in a plant defence response , 2001, The EMBO journal.

[6]  J.-H. Sheen,et al.  Ca2+-Dependent Protein Kinases and Stress Signal Transduction in Plants , 1996, Science.

[7]  Peter M. Gresshoff,et al.  Biology of plant-microbe interactions , 1996 .

[8]  Pierre Baldi,et al.  A Bayesian framework for the analysis of microarray expression data: regularized t -test and statistical inferences of gene changes , 2001, Bioinform..

[9]  Alok J. Saldanha,et al.  Java Treeview - extensible visualization of microarray data , 2004, Bioinform..

[10]  S. Luan.  The CBL-CIPK network in plant calcium signaling. , 2009, Trends in plant science.

[11]  Audrey Kauffmann,et al.  Bioinformatics Applications Note Arrayqualitymetrics—a Bioconductor Package for Quality Assessment of Microarray Data , 2022 .

[12]  Marie Boudsocq,et al.  Signaling specificity and complexity of MAPK cascades in plant innate immunity , 2008 .

[13]  Stan Pounds,et al.  False discovery rate paradigms for statistical analyses of microarray gene expression data , 2007, Bioinformation.

[14]  E. Lam,et al.  Phytotoxicity and Innate Immune Responses Induced by Nep1-Like Proteins[W] , 2006, The Plant Cell Online.

[15]  Wei Ma,et al.  The grateful dead: calcium and cell death in plant innate immunity , 2007, Cellular microbiology.

[16]  Y. Uno,et al.  Identification of proteins that interact with catalytically active calcium-dependent protein kinases from Arabidopsis , 2009, Molecular Genetics and Genomics.

[17]  Da-Peng Zhang,et al.  Two Calcium-Dependent Protein Kinases, CPK4 and CPK11, Regulate Abscisic Acid Signal Transduction in Arabidopsis[W] , 2007, The Plant Cell Online.

[18]  Mayte Suárez-Fariñas,et al.  Harshlight: a "corrective make-up" program for microarray chips , 2005, BMC Bioinformatics.

[19]  Jonathan D. G. Jones,et al.  A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence , 2007, Nature.

[20]  T. Boller,et al.  A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. , 2009, Annual review of plant biology.

[21]  G. Felix,et al.  NPP1, a Phytophthora-associated trigger of plant defense in parsley and Arabidopsis. , 2002, The Plant journal : for cell and molecular biology.

[22]  G. Church,et al.  Preferred analysis methods for Affymetrix GeneChips revealed by a wholly defined control dataset , 2005, Genome Biology.

[23]  Marie Boudsocq,et al.  Identification of Nine Sucrose Nonfermenting 1-related Protein Kinases 2 Activated by Hyperosmotic and Saline Stresses in Arabidopsis thaliana* , 2004, Journal of Biological Chemistry.

[24]  A. Harmon,et al.  Plants, symbiosis and parasites: a calcium signalling connection , 2005, Nature Reviews Molecular Cell Biology.

[25]  A. Reddy,et al.  Ca2+/calmodulin regulates salicylic-acid-mediated plant immunity , 2009, Nature.

[26]  S. Akira,et al.  Pathogen Recognition and Innate Immunity , 2006, Cell.

[27]  Cheng Li,et al.  Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application , 2001, Genome Biology.

[28]  F. Ausubel,et al.  MAP kinase signalling cascade in Arabidopsis innate immunity , 2002, Nature.

[29]  T. Boller,et al.  Perception of the Bacterial PAMP EF-Tu by the Receptor EFR Restricts Agrobacterium-Mediated Transformation , 2006, Cell.

[30]  D. Klessig,et al.  A Harpin Binding Site in Tobacco Plasma Membranes Mediates Activation of the Pathogenesis-Related Gene HIN1 Independent of Extracellular Calcium but Dependent on Mitogen-Activated Protein Kinase Activity , 2001, Plant Cell.

[31]  M. Cho,et al.  Calmodulin interacts with MLO protein to regulate defence against mildew in barley , 2002, Nature.

[32]  Jonathan D. G. Jones,et al.  Resistance Gene-Dependent Activation of a Calcium-Dependent Protein Kinase in the Plant Defense Response , 2000, Plant Cell.

[33]  J. Alonso,et al.  T-DNA mutagenesis in Arabidopsis. , 2003, Methods in molecular biology.

[34]  T. Speed,et al.  Summaries of Affymetrix GeneChip probe level data. , 2003, Nucleic acids research.

[35]  S Miyano,et al.  Open source clustering software. , 2004, Bioinformatics.

[36]  L. Piater,et al.  Innate immunity in plants and animals: striking similarities and obvious differences , 2004, Immunological reviews.

[37]  Yu-Chang Tsai,et al.  Innate Immunity Signaling: Cytosolic Ca2+ Elevation Is Linked to Downstream Nitric Oxide Generation through the Action of Calmodulin or a Calmodulin-Like Protein1[W][OA] , 2008, Plant Physiology.

[38]  R. Ranjeva,et al.  Proteinaceous and oligosaccharidic elicitors induce different calcium signatures in the nucleus of tobacco cells. , 2005, Cell calcium.

[39]  Robert Nadon,et al.  Comparison of small n statistical tests of differential expression applied to microarrays , 2009, BMC Bioinformatics.

[40]  F. Ausubel,et al.  Resistance to Botrytis cinerea Induced in Arabidopsis by Elicitors Is Independent of Salicylic Acid, Ethylene, or Jasmonate Signaling But Requires PHYTOALEXIN DEFICIENT31[W] , 2007, Plant Physiology.

[41]  T. Hartung,et al.  Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[42]  John N. Weinstein,et al.  Quality assessment of microarrays: Visualization of spatial artifacts and quantitation of regional biases , 2005, BMC Bioinformatics.

[43]  G. Felix,et al.  Bacteria-derived Peptidoglycans Constitute Pathogen-associated Molecular Patterns Triggering Innate Immunity in Arabidopsis* , 2007, Journal of Biological Chemistry.

[44]  Marie Boudsocq,et al.  Stress Signaling II: Calcium Sensing and Signaling , 2009 .

[45]  Jennifer L. Nemhauser,et al.  Different Plant Hormones Regulate Similar Processes through Largely Nonoverlapping Transcriptional Responses , 2006, Cell.

[46]  Rafael A Irizarry,et al.  Exploration, normalization, and summaries of high density oligonucleotide array probe level data. , 2003, Biostatistics.

[47]  P. Schulze-Lefert,et al.  A Glucosinolate Metabolism Pathway in Living Plant Cells Mediates Broad-Spectrum Antifungal Defense , 2009, Science.

[48]  Jonathan D. G. Jones,et al.  Bacterial disease resistance in Arabidopsis through flagellin perception , 2004, Nature.

[49]  J. Sheen,et al.  Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis , 2007, Nature Protocols.

[50]  Frederick M. Ausubel,et al.  Glucosinolate Metabolites Required for an Arabidopsis Innate Immune Response , 2009, Science.

[51]  Filip Rolland,et al.  A central integrator of transcription networks in plant stress and energy signalling , 2007, Nature.

[52]  Wenzislava Ckurshumova,et al.  A high-throughput chemical screen for resistance to Pseudomonas syringae in Arabidopsis. , 2008, The Plant journal : for cell and molecular biology.

[53]  S. Rhee,et al.  MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. , 2004, The Plant journal : for cell and molecular biology.

[54]  P. Zimmermann,et al.  Gene-expression analysis and network discovery using Genevestigator. , 2005, Trends in plant science.

[55]  J. Sheen,et al.  Calcium Signaling through Protein Kinases. The Arabidopsis Calcium-Dependent Protein Kinase Gene Family1 , 2002, Plant Physiology.

[56]  R. Ranjeva,et al.  Calcium in plant defence-signalling pathways. , 2006, The New phytologist.

[57]  R. Tibshirani,et al.  Significance analysis of microarrays applied to the ionizing radiation response , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[58]  T. Boller,et al.  Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. , 1999, The Plant journal : for cell and molecular biology.

[59]  H. Yoshioka,et al.  Calcium-Dependent Protein Kinases Regulate the Production of Reactive Oxygen Species by Potato NADPH Oxidase [W][OA] , 2007, The Plant Cell Online.

[60]  G. Martin,et al.  Specific Bacterial Suppressors of MAMP Signaling Upstream of MAPKKK in Arabidopsis Innate Immunity , 2006, Cell.